NO152937B - PROCEDURE FOR THE PREPARATION OF AN ATENENUATED PANELUCOPENIA VIRUS - Google Patents

Abstract

PROCEDURE FOR PREPARING AN ATTENUATED PANLEUKOPENI VIRUS.

Description

The invention relates to a method for the production of attenuated Panleukopenia virus strain B W 10 3 which is deposited in the Institut fiir Hygiene und Epidemiologie in Prague no. CNTCC

AO 3/77.

Feline distemper is a felid-specific viral disease. Producing-

one, a parvovirus, has an electron microscopic size of 20 - 25 nm. The virus is transmitted via the respiratory and digestive tract, via nasal secretions and faeces it is excreted. After an incubation period of 2 to 6 days, the disease begins with refusal, fever, vomiting, mostly diarrhea and increasing weakness with dehydration due to fluid loss. A high degree of panleukopenia appears as a characteristic. The lethality is up to 90%.

Cats that survive the disease become immune. They can transfer antibodies to young cats via colostral milk. However-

time they are also soon without protection, as the maternal antibodies gradually degrade.

Feline plague is widespread worldwide, the infectivity and pathogenicity of its agents is very high and cats are therefore very threatened.

Attempts have already been made to extract an attenuated virus suitable for the production of a cat plague vaccine by passage in tissue cultures. However, at least six passages, especially ten to thirty passages, are not sufficient to obtain a stable attenuated virus that can serve as the active principle of the vaccine. It is observed that the vaccination virus is secreted and absorbed by other cats. During such animal crossings, back mutations can occur.

Embryonic tissue from cats has also already been used for propagation of the attenuated feline plague virus. However, the cat embryo is very little tissue, so that this method

offered for financial reasons.

The invention relates to a method for production

of an attenuated panleukopenia virus strain B W 103 which is deposited in the Institut fur Hygiene und Epidemiologie in Prague under no. CNTCC AO 3/77, the method being characterized by passing pathogenic panleukopenia virus at least 80 times on cat kidney cells, and then at least 18 times

on permanent tissue cells from felids or mustelids, preferably on Crandell's cat kidney cell line.

The first step in the attenuation, which suitably comprises 80 to 100 passages, takes place in cell cultures of cat kidneys, which are cultivated in a normal nutrient medium for cell propagation. The culture of the cells can take place both as a cell lawn and as a cell suspension. A temperature of 35 to 39°C, preferably 36.5 to 37.5°C, must be observed. Such a culture must be infected early, however at the latest when the cells have grown to approx. 75%. The excess of the previously infected cell culture serves as the infection medium.

trip. It is added to the new cell culture in a ratio of 1:5 to 1:120. 2 to 16 days after infection, the supernatant can be recovered and transferred to the next cell culture.

After 80 to 100 passages in the mentioned manner, the virus-containing supernatant is transferred to a permanent tissue cell culture of felids or mustelids. Corresponding permanent cultures are known from the literature. An example of such permanent cell cultures is Crandell's cat kidney cell line, a permanent cat kidney cell. The infection of this cell culture with the supernatant from an infected cell culture and the further processing takes place as previously discussed.

After at least 18 passages in permanent tissue cells, the virus is sufficiently attenuated. It is completely apathogenic, harmless and well compatible. During the attenuation, it does not lose its immunogenicity and the vaccine produced from it has good immunizing properties.

The attenuated virus, which has the scientific designation "panleukopenia virus, strain B'W 103", was deposited in the Institute of Hygiene and Epidemiology, Prague, under registration number CNTCC AO 3/77.

The thus attenuated virus can be propagated in tissue cultures of cat fetuses. Fetuses that were removed in the stage of organogenesis in approximately the last third of pregnancy are particularly suitable for this. For the establishment of tissue cultures, the fetuses can be processed as a whole or parts thereof. It is not profitable for the cell culture to use extremities, the head and the skin. The cells are processed in the usual way, e.g. by trypsinization established as a stationary culture, as a rolling culture or as a suspension culture and infected as previously discussed with the attenuated virus for propagation. The harvest of the above takes place after 2 - 6 days. The infected cell cultures can be further cultivated with the addition of a new nutrient solution. This operation can be repeated several times, e.g. with the stationary culture at least 4-6 times and with the rolling culture at least 20-25 times. Then the cells are generally depleted. In the final processing, the cells for the production of a larger yield can be destroyed, for example, by deep freezing or ultrasonic treatment.

That achieved by the processing of the breeding culture

material can be processed in the usual way into a grafting material. It pays to add a stabilizer, for example gelatin, meat extract, peptone or sugar solutions, especially when lyophilization of the grafting material is prescribed. Such a one pays off because of the better durability. However, the grafting material can also be used as a liquid grafting material. The addition of one of the usual adjuvants is possible if desired.

The grafting substance can - if it is available in lyophilized form, after dissolution in one of the usual solvents for this purpose - administered subcutaneously or intramuscularly. It has

compared to known grafting substances the following advantages:

1) Great purity of the antigens.

2) When multiplying on homologous tissue, no foreign egg white reactions. With repeated administration, no shock reactions or allergies. 3) Consistent quality and purity of the inoculum virus, as propagation takes place on a uniformly tested cell substrate.

4) Fast protection.

Panleukopenia living inoculum was

examined in a number of investigations and tests in the laboratory and in animal experiments for properties, tolerability, harmlessness and effectiveness.

Experiments and results:

Harmlessness.

Tests on harmlessness took place on white mice and guinea pigs. 5 mice are inoculated subcutaneously with each 0.5 ml of the reconstituted vaccine. 2 guinea pigs each receive 2 ml intraperitoneally. During an observation period of 10 days, the experimental animals must remain healthy. Special value was placed on harmlessness and tolerability testing in cats. In controlled laboratory and field trials, the inoculum was subcutaneously or intramuscularly inoculated into 962 predominantly isolated cats and 174 big cats, such as lions, tigers, cheetahs, etc.

and usually observed for 14 days, sometimes several weeks.

After the control of local and general reactions, a leukocyte count is also carried out in laboratory tests. Thereby, it turned out that the inoculant was well tolerated. Also the inoculation of SPF (SPF = specified pathogen free) - cats, which were partially kept in isolation over 2 years, gave no cause for regret.

Stability

For a consistently good effect of the inoculant according to the invention, a good durability is necessary in addition to the virus titer. The examination of this took place by determining the content of inoculum virus after storage of the inoculum material at different temperatures for up to 24 months after production. The achieved antigen titers appear in table 1.

Effect and tolerability

Alongside examination of the content of live inoculum virus by virus titration in cell culture, the product according to the invention was examined in a series of immunization trials on cats. The greater prevalence of panleukopenia virus and other felidpathogenic agents must be taken into account when carrying out the experiments on purchased cats in corresponding quarantine holdings. In order to avoid foreign infections and to achieve accurate test results, it is appropriate to use a specified pathogen-free cat, so-called SPF cat.

Animal testing No. 1.

In a first animal experiment, the effect and tolerability of the vaccine was investigated on 6 SPF cats. Of the cats housed together in an isolation facility, 4 were vaccinated subcutaneously with the inoculant. 2 cats remained as unvaccinated controls. In addition to an examination of the general state of health, special attention was paid to whether, after administration of the inoculant, there was a noticeable decrease in the leukocyte value compared to a non-inoculated cat. This was not the case in the present and further experiments. The effect was examined serologically by antibody measurement and subsequent challenge infection. The results of this examination are summarized in table 2. From this it can be seen that before the inoculation the cats were sensitive to panleukopenia. 2 weeks later, the vaccinated cats were already serologically immune and susceptible, the controls, on the other hand, remained unprotected and got infected.

Animal testing No. 2.

Inoculum containing live apathogenic panleukopenia viruses usually results in rapid graft protection,

which is above all to attribute the phenomenon of interference. This prevents the propagation of pathogenic virus in the host cell by grafting virus. In an experiment on 12 young cats, it was investigated whether these already 72 hours after administration of the inoculant are sufficiently protected against a stress infection. The results of these investigations are reproduced in table 3. The trial shows that the inoculum is able to protect against panleukopenia in a short time, namely within 3 days. All the cats were unprotected before vaccination.

3 days later, the inoculated animals already resisted a challenge infection with pathogenic panleukopenia virus. In the case of a daily leukocyte count carried out after the challenge infection, which extended over two weeks, it was established that the cats remained healthy in appearance and did not show a clear leukocyte drop. The baseline and minimum values established during the examination were entered in table 3. A leukocyte count below 2000/mm 3 is usually a characteristic sign of panleukopenia. The non-vaccinated control cats became more or less severely ill with panleukopenia, with 4 out of 6 animals becoming infected.

Animal testing No. 3.

According to animal experiments 2, the inoculant produced from

the weakened virus already 3 days after vaccination (3 d.p.v.) for a disease of panleukopenia (PL).. For

to determine whether the same vaccine also already before the 3rd d.p.v. provides a protection against PL, the following experiments were carried out.

16 panleukopenia-susceptible cats were vaccinated with each 1 dose of the inoculum and divided into 4 groups of 4 animals each. A group of these were already immediately after the vaccination

in other groups in succession each time at a distance of 1 day charged with pathogenic panleukopenia virus, six untreated control cats were infected together with the last group on 3 d.v.p. As table 4 shows, this experiment shows that the inoculant already one day after vaccination manages to protect a part of the vaccinated cats from a disease of panleukopenia. Of four cats of the group in question, only one animal became ill and died of panleukopenia, the other three animals remained healthy.

Of the animals that were challenged two days after the vaccination, only one of four cats on the 4th and 5th day after the infection showed a small leukocyte drop, (to 4200 or 5100/mm<3>). The cats infected three days after vaccination withstood the challenge without reaction.

Of the control cats, all died of panleukopenia.

Further details can be derived from Table 4.

Animal testing No. 4.

To investigate the duration of protection, a further trial was carried out on SPF cats that were kept under strict isolation. Out of 10 SPF cats, 8 each received a dose of the inoculant subcutaneously. The two non-vaccinated control cats were examined for excretion and possible further processing of the inoculated virus or PL virus introduction placed in the same room. Blood samples were taken for antibody measurement in all cats. The results of this investigation appear in table 5. The trial shows that the inoculant leads to an active immunity with a high-quality antibody that gives vaccinated cats, over the overall observation period, acceptable protection against panleukopenia. It also shows that the control cats remain antibody-free. This means that during the trial period there was neither a further spread of the inoculation virus nor an introduction of PL viruses, which at the inoculations would have led to a natural breeding, resp. the controls to a pan-leukopenia disease with subsequent antibody development. When the experimental cats were cared for and observed daily, it could be established that during the total period of the experiment no reactions occurred which would suggest intolerance or harm. Pathogenic panleukopenia virus isolated from a cat that had died of panleukopenia was used to infect a cat that was euthanized 7 days later in a severely ill state with a leukocyte count of 200 mm 3 .

The kidneys were removed and treated in a 25% trypsin solution to extract a cell suspension. The formed virus suspension is sedimented and the sediment is suspended in a ratio of 1:300 in a culture medium of the following composition:

52.2% Hanks' medium

5.8% lactalbumin hydrolyzate

20.0% Tissue Culture Medium 199

20.0% calf serum

1.0% sodium bicarbonate (5%-ig)

1.0% NSP solution.

The suspension is kept at 37°C, thereby causing the panleukopenia virus to multiply in the cells. The virus can be detected by transferring the supernatant culture to healthy cats.

The further cultivation of the virus takes place as follows: From healthy cats, the kidneys are removed and trypsinized in the above-mentioned manner and suspended in the nutrient solution. When the cell mass has grown to approx. 75%, it is inoculated with the virus-containing supernatant from the panleukopenia virus culture.

To this, the above is mixed with fresh nutrient medium

in a ratio of 1:10 and poured over the cells. After four days, the supernatant containing the multiplied panleukopenia virus is decanted. After approx. After 80 repetitions, the cat kidney cells used until then are replaced by a permanent cat kidney cell line according to Crandell and the passages are repeated a further 17 times.

The following medium is used for the cultivation of stable cat kidney cells:

10% Eagle's Medium

10% calf serum

1.5% sodium bicarbonate (5%-ig)

1.0% NSP solution

ad 100% Aqua bidest.

With virus inoculation, serum formation is reduced to 3%. After the 103rd passage, the virus has lost its pathogenicity, but retains its good immunizing properties. It can be used for the production of an inoculant.

Claims (1)

Process for the production of an attenuated panleukopenia virus strain BW 103 which is deposited in the Institut fur Hygiene und Epidemiologie, in Prague, under No. CNTCC AO 3/77, characterized by passing pathogenic panleukopenia virus at least 80 times on cat kidney cells and then at least 18 times on permanent tissue cells from felids or mustelids, preferably on Crandell's cat kidney cell line.